犬成年骨骼肌成肌细胞的培养纯化、鉴定及生长特性的研究

目的建立简便、有效的成年犬骨骼肌成肌细胞（skeletal myoblast cells，SMCS）体外分离培养、纯化和鉴定的方法，并观察犬SMCs的生长特性。方法采用机械分解法与IA型胶原酶和胰蛋白酶的二步酶消化法相结合，分离消化法培养获取犬SMCs，用DMEM培养液培养，以Ficoll梯度液纯化，进行Desmin免疫组化鉴定，并观察细胞形态、生长特性。结果犬SMCs培养后经台盼蓝检查成活率达95％以上，在接种后4～5d增殖达高峰，细胞生长旺盛；Fiooll分离纯化的SMCs纯度较高；Desmin免疫细胞化学染色有97％的SMCs胞浆呈阳性反应。结论改良方法获得的SMCs具有良好的增殖、分化能力；采用FicollU纯化以血清培养，即可获得高产量高纯度的SMCs。

Culture, Purification, Identification and Proliferative Characteristics of Adult Canine Skeletal Myoblast Cells

Objective Establish a thrifty, convenient and practical procedure of the culture, purification, identification and observe the prolif erative characteristics of adult canine skeletal myoblasts cells ( SMCs) for cellular cardiomyoplasty in vitro. Methods Adult canine SMCs were obtained using the method of two - step digestion (inclduing collagenase IA and trypsin) combined with the method of mechanical cell dissociation, and purified with Ficoll gradient liquid. Morphorlogi-cal characteristics and growth curve were observed; The SMCs were identified by cellular immunochemical staining which myoblast were tested by Desmin immunohisto - chemistry stains. Results Adult canine SMCs obtained by the modified method replicated very fast and formed myotubes in differential medium. The SMCs viability is up to 95% by Trypan blue, and the cells proliferation arrived climax during 4~5 day after planted. The SMCs purity is up to 97% by Desmin immunohistochemical stains test. Conclusion The modified method can be used as a thrifty and convenient choice for getting adult canine SMCs, which could be purified by Ficoll and cultured with DMEM culture liquid contained serum in high quantity.